The present invention concerns a novel method to analyze identity, protein and/or peptide pattern and as well the stability of samples containing physiologically acceptable enzyme mixtures with lipolytic, proteolytic and amylolytic activity, but especially of mixtures of digestive enzymes such as pancreatin, in particular in the context of the manufacture of medicinal products comprising said enzyme mixtures, e.g. precipitated pancreatin or pancreatin mini-microspheres.
It is the object of the invention to provide new analytical method suitable for pharmaceutical preparations comprising mixtures of digestive enzymes such as pancreatin, in particular also in the context of the manufacture of medicinal products comprising said enzyme mixtures, e.g. pancreatin or pancreatin mini-microspheres. In particular it is the object to provide an analytical method suitable and reliable to be validated for pharmaceutical manufacturing for analyzing and determining the identity, protein and/or peptide pattern and as well the stability of said digestive enzyme samples. A further object is to provide said analytical method in conditions which are optimized for the analysis of pancreatin, in particular precipitated pancreatin or pancreatin mini-microspheres samples.
According to the invention, physiologically acceptable enzyme mixtures with lipolytic, proteolytic and amylolytic activity, such as suitable enzyme mixtures of microbial origin and/or especially mixtures of digestive enzymes of animal origin such as preferably pancreatin or pancreatin-like mixtures of digestive enzymes, are analyzed according to the analytical methods essentially described in this patent specification.
For the present invention, physiologically acceptable enzyme mixtures with lipolytic, proteolytic and amylolytic activity can be analyzed that are of any animal or microbiological origin. The enzyme mixtures with lipolytic, proteolytic and amylolytic activity analyzed by the method of the invention can be both of purely microbial origin, purely animal origin or may also be a mixture of enzymes of animal and microbial origin.
In one variant of the invention, therefore, the enzyme mixture used is of purely microbial origin. Especially enzymes produced by bacteria, i.e. by the Bacillus or Pseudomonas strains, or by fungal cultures such as molds, for example of the Rhizopus and Aspergillus strains, are especially suitable as microbial enzymes. Examples of such physiologically acceptable bacterial and/or mold fungi enzymes are already described in the state of the art, e.g. in connection with their synthesis and use for the treatment of maldigestion. Lipases may be derived from, for example, Bacillus or Pseudomonas strains, amylases and lipases from mold fungi, for example of the Rhizopus strain, and proteases, for example, also from Aspergillus. 
One preferred variant of the invention, however, will involve the use of mixtures of digestive enzymes with lipolytic, proteolytic and amylolytic activity that in their properties closely resemble pancreatin. For the present invention, mixtures of digestive enzymes containing pancreatin and especially pancreatin itself are preferably used, and one or more microbial enzymes, i.e. enzymes synthesized by microorganisms, of the group of lipases, proteases and amylases may if desired be added to the pancreatin or the mixtures of digestive enzymes containing pancreatin. Most preferred the method according to the invention is suitable for analysis of precipitated pancreatin or pancreatin mini-microspheres samples.
Pancreatin is a known enzyme mixture with lipolytic, proteolytic and amylolytic activity which is available for example, under the trade name Creon®, in the form of granules, pellets or capsules containing enteric coated microspheres and is used medically for enzyme replacement, for example in pancreatic insufficiency, digestive insufficiency after stomach operations, liver and biliary diseases, cystic fibrosis and chronic pancreatitis. Pancreatin is generally obtained as a mixture of natural enzymes by extraction from porcine pancreas, for example according to the process described in U.S. Pat. No. 4,019,958 (=DE 25 12 746) and German patent no. DE 42 03 315, and is then converted into the desired galenical form in a manner known to the art. The pancreatic enzymes are usually administered orally in the form of solid preparations.
In one variant of the invention, the pharmaceutical preparations to be analyzed in accordance with the invention contain preferably pancreatin or mixtures of digestive enzymes containing pancreatin. These pharmaceutical preparations analyzed according to the invention can contain pancreatin or mixtures of digestive enzymes containing pancreatin and possibly in addition to pancreatin one or more physiologically acceptable enzymes from the group of lipases, proteases and amylases, of the kind that can be obtained from microorganisms. Microbial enzymes used in this supplement include especially the bacterially synthesized enzymes already mentioned above, for example by the Bacillus or Pseudomonas strains, or by fungal cultures such as mold fungi, for example of the Rhizopus or Aspergillus strains. The lipases contained in addition to the pancreatin or the mixtures of enzymes containing pancreatin may originate, for example, from Bacillus or Pseudomonas strains, added amylases and lipases from mold fungi, for example of the Rhizopus strain, and added proteases, for example, also from Aspergillus. 
It has now been found that the physiologically acceptable enzyme mixtures with lipolytic, proteolytic and amylolytic activity such like pancreatin or non-animal sourced enzyme mixtures which can be obtained from microbial and/or animal sources and described with reference to this invention can be analyzed very efficiently according to the methods of the present invention. The invention provides a powerful and reliable (reproducible) method for e.g. analyzing and determining the identity, protein and/or peptide pattern and as well the stability of said digestive enzyme compositions or samples containing physiologically acceptable enzyme mixtures with lipolytic, proteolytic and amylolytic activity. It is evident to the skilled artisan that he may vary given parameters to a certain extent without loosing the overall functionality of the method according to the present invention; e.g. it may be wished to adapt the parameters indicated for performing the method in the following description, the examples, Tables and Figures by +/−10%, in particular by +/−5%.
Thus, the invention pertains to an analytical method for characterization and/or specification of protein samples containing physiologically acceptable digestive enzyme mixtures with lipolytic, proteolytic and amylolytic activity, which are used in the manufacture of pharmaceutical preparations for the treatment of disorders and/or disorders, by two-dimensional gel electrophoresis (2D GE), said method comprising:                (a) sample preparation by solving of an enzyme mixture sample in a solvent composition for gel electrophoresis comprising a specified solvent suitable to solve protein materials, an internal standard for quantification of proteins, and a protease inhibiting agent;        (b) an isoelectrical focussing step for defining the first dimension of the gel electrophoresis and applying a gradient for separation of the protein fractions;        (c) a subsequent pre-treatment step comprising re-buffering;        (d) transfer to the second dimension and separation by SDS-PAGE;        (e) fixing and staining of the gels resulting from step (d); and        (f) densitometrical evaluation by fluorescence scanning.        
The two-dimensional gel electrophoresis method is particularly suited for analyzing and determining the identity, protein and/or peptide pattern and as well the stability of said digestive enzyme compositions or samples containing physiologically acceptable enzyme mixtures with lipolytic, proteolytic and amylolytic activity, with molecular weights of the protein or peptide fractions from about 8 kDa (kilo Dalton) and above. In a variant the invention is particularly suited for analyzing and determining the identity, protein and/or peptide pattern and as well the stability of pancreatin, and in particular of precipitated pancreatin or pancreatin mini-microspheres. Parameters applicable in performing the method variants of the present invention are detailed below in the sections of the description pertaining to “Identification of spots using MALDI-TOF MS”, “Stress Test Study for Precipitated Pancreatin”, “Analytical Procedure for Determining Identity and Protein Pattern of Precipitated Pancreatin Samples by Two-Dimensional Gel Electrophporesis”, and related Tables, and are further illustrated by the Figures given in the context of this invention.
For digestive enzyme samples, e.g. pancreation and in particular precipitated pancreatin or pancreatin mini-microspheres, with molecular weights of the protein or peptide fractions below about 8 kDa (kilo Dalton) the method may be supplemented according to a variant of the invention by additional application of analytical RP-HPLC method.
In an aspect the invention as defined above pertains to analysis of an enzyme mixture of microbially synthesized lipases, proteases and amylases. In another aspect the invention as defined above pertains to analysis of a pancreatin and/or a pancreatin-like mixture of digestive enzymes. In yet another aspect the invention as defined above pertains to analysis of a pancreatin a sample which is a precipitated pancreatin or a pancreatin mini-microspheres.
In a further aspect the invention as defined above pertains to an analytical method, wherein the solvent used in step (a) to dissolve the sample is a lysis buffer of 7M urea, 2M thiourea, 4% (w/v) CHAPS, 1% (w/v) DTT, and 0.5% Pharmalyte® at pH 3-10.
In a yet further aspect the invention as defined above pertains to an analytical method, wherein the internal standard for quantification of proteins used in step (a) is phosphorylase B, preferably rabbit phosphorylase B, or carbonic anhydrase, preferably bovine carbonic anhydrase.
In an additional aspect the invention as defined above pertains to an analytical method, wherein the protease inhibiting agent is Mini Complete and/or Pefabloc.
In a further aspect the invention as defined above pertains to an analytical method, wherein the solvent used in step (a) to dissolve the sample is Lp3 composed of 1.5 mg Mini Complete dissolved in 2 ml lysis buffer of 7M urea, 2M thiourea, 4% (w/v) CHAPS, 1% (w/v) DTT, and 0.5% Pharmalyte® at pH 3-10; and: 1 mg Pefabloc dissolved in 2 ml lysis buffer; in a ration 1:1 v/v.
The invention as defined above pertains also to an analytical two-dimensional gel electrophoresis method, wherein said method is applied for the characterization and quantification of protein and/or peptide fractions with a molecular weight above about 8 kD.
In a further aspect the invention as defined above pertains to an analytical method, which comprises determining the identity and/or the protein and/or peptide pattern of pancreatin, preferably of a precipitated pancreatin sample or of a pancreatin mini-microspheres sample.
In another aspect the invention as defined above pertains to an analytical method, which comprises the identification of protein and/or peptide spots using in addition MALDI-TOF-MS.
In one aspect the invention as defined above pertains to an analytical method, which is performed as a stress or stability test for determining the identity and/or the protein and/or peptide pattern of pancreatin, preferably of a precipitated pancreatin sample or of a pancreatin mini-microspheres sample, and impurities and/or degradants, and optionally comprising also the quantification said proteins, peptides, impurities and /or degradants.
In another aspect the invention as defined above pertains to an analytical method, wherein said method further comprises the characterization and quantification of low molecular weight protein and or peptide fractions with a molecular weight below about 8 kD by RP-HPLC.
In still another aspect the invention as defined above pertains also to a solvent composition suitable for characterization and/or specification of a sample of physiologically acceptable enzyme mixtures with lipolytic, proteolytic and amylolytic activity, which are used in the manufacture of pharmaceutical preparations for the treatment of disorders and/or disorders, by two-dimensional gel electrophoresis, comprising    (a) as solvent suitable for gel electrophoresis and to solve protein materials which solvent is a lysis buffer of 7M urea, 2M thiourea, 4% (w/v) CHAPS, 1% (w/v) DTT, and 0.5% Pharmalyte® at pH 3-10;    (b) an internal standard for quantification of proteins; and    (c) and a protease inhibiting agent.
In a variant of this solvent, the is a solvent composition, wherein the solvent to dissolve the sample is Lp3 composed of 1.5 mg Mini Complete dissolved in 2 ml lysis buffer of 7M urea, 2M thiourea, 4% (w/v) CHAPS, 1% (w/v) DTT, and 0.5% Pharmalyte® at pH 3-10; and: 1 mg Pefabloc dissolved in 2 ml lysis buffer; in a ration 1:1 v/v.
List of Some Abbreviations and/or Terms Used in the Following:
mmsmini-microspheres (pancreatin mini-microspheres)HClhydrochloric acidAPIactive pharmaceutical ingredientNDANew Drug ApplicationFDAFood and Drug AdministrationMALDI-TOF MSmatrix assisted laser desorption and ionizationmass spectroscopyUTLIEFultrathin-layer isoelectric focusingESI-MSelectrospray ionization mass spectroscopy
The analytical methods according to the invention, in particular after validation for pharmaceutical and regulatory purposes, are preferably intended to be used for characterization and specification setting of precipitated pancreatin and may also be applied to pancreatin enteric-coated minimicrospheres (pancreatin mini-microspheres).
For example, a product specification to be filed for the NDA for the active ingredient precipitated pancreatin and the dosage form pancreatin enteric-coated mms covers the items identification, purity, assay, gastric juice resistance and release of enzymes. State of the art identification is based on enzymatic assays which are used for determining the activity of the enzymes in both, the API and the dosage form. “Purity” also includes the determination of residual solvents (API, mms), fat (API), water (API, mms) and microbiological quality. For taking into account current FDA requirements and expectations based on Q6B Guidance “Specifications, Test Procedures and Acceptance Criteria for Biotechnological/Biological Products” more detailed characterization is regarded necessary for the drug substance and the dosage form with special attention to identification and quantification of different classes of enzymes, impurities and degradants from these enzymes. Results and methods from characterization will be selected for specification setting.
Therefore the present invention proposes for characterization and specification setting the use of two-dimensional gel electrophoresis (2D GE), because it was found that, since precipitated pancreatin is a complex mixture of different classes of constituents, two-dimensional gel-electrophoresis is expected to give by far the greatest selectivity for separation of peptides and proteins, i.e. different classes of enzymes, impurities and degradants of proteins. Furthermore, imaging of stained gels permits quantification of the constituents and comparison of the protein and/or peptide patterns in pancreatin samples, samples of precipitated pancreatin or pancreatin mini-microspheres. The present invention shows that identification of the most prominent spots can be performed by spot-picking and MALDI-TOF MS after tryptic digest.
In general, the separation by the two-dimensional gel electrophoresis method according to the present invention will be performed in the first dimension (step (b) isoelectric focussing) from aqueous buffered solutions of pancreatin samples or mms samples, after desalting of the sample, on gels with a pH gradient from 3 to 10 to cover a broad range of potential constituents or compounds. Focussing is performed on immobiline dry strips. An exemplary gradient to start with is tabulated below:
Voltage [V]Step (2D GE isoelectric focussing)up to abooutTime [h] up to about11504, preferably 123004, preferably 136003, preferably 1412001524001635008, preferably 7.25
In general, the separation in the second dimension (step (d) SDS-PAGE) of the two-dimensional gel electrophoresis method according to the present invention will be performed on hand-made gels (for example under following condition:T=13%, C=3%) with e.g. a SDS-GLYCIN-TRIS buffer with an exemplary gradient as tabulated below:
Current [mA]Stepabout e.g.Voltage [V] about e.g.180Max 452150Max 200310For security
In general, staining is performed after fixation with for example ethanol/acetic acid mixture with a fluorescent dye and subsequent destaining in for example ethanol/acetic acid. After washing with water, densitometric scanning is performed. Afterwards, staining with for example colloidal Coomassie blue is performed for identification by MALDI-TOF MS. For this purpose, spots will be picked from the gel and subjected to a tryptic digest. Peptides are eluted from the gel for example with acetonitrile/0.1% TFA and purification on a C18 ZipTip Column. After co-crystallization with 2,5-dihydroxy benzoic acid, the extracts are pipetted on the target plate.
As an example to illustrate the applicability and usefulness of the present invention in the analytical method three batches each of three species of precipitated pancreatin (glands from different countries and different manufacturing processes), including Pancreatin SPL 85 were selected. One sample of each batch was applied on a gel, for one batch of each species the analysis was performed threefold to check reproducibility of the precipitation step, the sample preparation and the separation. The spots were quantified and identification of characteristic spots was performed.
As an example to illustrate the applicability and usefulness of the present invention in stability testing, the same batches as used before to illustrate the applicability and usefulness were subjected to stress conditions (temperature, humidity, light) to determine any loss of activity and then to analyze or investigate also differences and, if applicable, identify potential degradants.
As indicated above, for digestive enzyme samples, e.g. pancreatin and in particular precipitated pancreatin or pancreatin mini-microspheres, with molecular weights of the protein or peptide fractions below about 8 kDa (kilo Dalton) the method may be supplemented according to a variant of the invention by additional application of analytical RP-HPLC method. Parameters applicable in performing the RP-HPLC method variants of the present invention are detailed below in the section of the description pertaining to “Feasibility of RP-HPLC with MALDI-TOF-MS for Analysis of Pancreatin”.
Identification of spots using MALDI-TOF MS”, “Stress Test Study for Precipitated Pancreatin”, “Analytical Procedure for Determining Identity and Protein Pattern of Precipitated Pancreatin Samples by Two-Dimensional Gel Electrophoresis”, and related Tables, and are further illustrated by the Figures given in the context of this invention.
HPLC is a widely automated, well reproducible, highly selective method which is widely used for routine analysis, also in protein analysis. Quantification of compounds is easy and identification of peaks can be performed by LC-ESI-MS. Peptides of lower molecular mass and other low-molecular compounds can be detected and identified so that the method is complementary to e.g. two-dimensional gel electrophoresis or SDS-PAGE. It can therefore be used in particular for fingerprinting, identification purposes and quantification of enzyme classes, impurities and degradants.
Usually the HPLC method involves for example an agilent HPLC-equipment consisting of: Autosampler G 1313A; Quat. pump G 1311A; UV-detector G 1314A; Vacuum degasser G 1322A; HP Column Oven G1316A; 1100 control module G 1323A; LAN-interface 35900E; and ChemServer; or an equivalent system. A typical HPLC column may be as an example a MODULO O-CART QS UPTISPHERE 5 WRP, Interchim (UP5WRP$15QS) with a stationary phase of RP 18, 5.0 μm, tubing material of stainless steel with a length of 150 mm and an internal diameter of 3.0 mm; or a comparable equivalent HPLC column. The RP 18, 5.0 μm phase is beneficial for example, as it is possible to operate with 100% water, and it is suitable for proteins and peptides. Further examples for suitable columns are e.g. Polaris 5 μm C18-A 150×4.6 mm obtainable from Varian B. V., Middelburg, The Netherlands (article order no. A2000150X046); or e.g. Cogent Bidentate, C( (Octyl), 4 μm, 300 A, 150×4.6 mm from MicroSolv Technology Corporation, Long Branch, N.J. 07740, USA.
The HPLC method may be operated under following exemplary conditions:
Operating modeGradient HPLCMobile phasemobile phase Awater/TFA 0.05% (v/v)mobile phase Bacetonitrile/TFA 0.05% (v/v)GradientTime [min]% A% B01000linear gradient to751090linear gradient to75.11000isocratic801000equilibrationFlow rate 1.0 ml/minPeriod of analysis 75 minTemperature 20 ± 5.0° C.Injection volume 10 μlFor detection, for example, a UV-detector may be used at a wavelength of 214 nm